Browsing by Author "Kaefer, Samuel L."

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    Early Changes in Porcine Larynges Following Injection of Motor-Endplate Expressing Muscle Cells for the Treatment of Unilateral Vocal Fold Paralysis
    (Wiley, 2024) Kaefer, Samuel L.; Zhang, Lujuan; Morrison, Rachel A.; Brookes, Sarah; Awonusi, Oluwaseyi; Shay, Elizabeth; Hoilett, Orlando S.; Anderson, Jennifer L.; Goergen, Craig J.; Voytik-Harbin, Sherry; Halum, Stacey; Otolaryngology -- Head and Neck Surgery, School of Medicine
    Objectives: No curative injectable therapy exists for unilateral vocal fold paralysis. Herein, we explore the early implications of muscle-derived motor-endplate expressing cells (MEEs) for injectable vocal fold medialization after recurrent laryngeal nerve (RLN) injury. Methods: Yucatan minipigs underwent right RLN transection (without repair) and muscle biopsies. Autologous muscle progenitor cells were isolated, cultured, differentiated, and induced to form MEEs. Three weeks after the injury, MEEs or saline were injected into the paralyzed right vocal fold. Outcomes including evoked laryngeal electromyography (LEMG), laryngeal adductor pressure, and acoustic vocalization data were analyzed up to 7 weeks post-injury. Harvested porcine larynges were examined for volume, gene expression, and histology. Results: MEE injections were tolerated well, with all pigs demonstrating continued weight gain. Blinded analysis of videolaryngoscopy post-injection revealed infraglottic fullness, and no inflammatory changes. Four weeks after injection, LEMG revealed on average higher right distal RLN activity retention in MEE pigs. MEE-injected pigs on average had vocalization durations, frequencies, and intensities higher than saline pigs. Post-mortem, the MEE-injected larynges revealed statistically greater volume on quantitative 3D ultrasound, and statistically increased expression of neurotrophic factors (BDNF, NGF, NTF3, NTF4, NTN1) on quantitative PCR. Conclusions: Minimally invasive MEE injection appears to establish an early molecular and microenvironmental framework to encourage innate RLN regeneration. Longer follow-up is needed to determine if early findings will translate into functional contraction.
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    High-Riding Innominate Artery: Booby Trap for ICU Tracheotomy
    (Wiley, 2025) Ramakrishnan, Vijay R.; Kaefer, Samuel L.; Otolaryngology -- Head and Neck Surgery, School of Medicine
    Despite tracheotomy being a routine procedure, it is not rare to encounter anatomic irregularities that can compromise its success. In this report, we describe a case in which a high riding innominate artery was identified within the surgical trajectory moments before incision, which ultimately necessitated airway securement using an alternative laryngological procedure.
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    Neuromuscular junction visualization in paraffin‐embedded thyroarytenoid muscle sections: Expanding options beyond frozen section analysis
    (Wiley, 2025-01-07) Kaefer, Samuel L.; Shay, Elizabeth O.; Morrison, Rachel A.; Zhang, Lujuan; Voytik-Harbin, Sherry; Halum, Stacey; Otolaryngology -- Head and Neck Surgery, School of Medicine
    Objectives: The current gold standard for immunofluorescent (IF) visualization of neuromuscular junctions (NMJs) in muscle utilizes frozen tissue sections with fluorescent conjugated antibodies to demarcate neurons and IF alpha-bungarotoxin (α-BTX) to demarcate motor endplates. Frozen tissue sectioning comes with inherent inescapable limitations, including cryosectioning artifact and limited sample shelf-life. However, a parallel approach to identify NMJs in paraffin-embedded tissue sections has not been previously described. Methods: Yucatan minipig thyroarytenoid (TA) muscle was harvested and prepared as 5-μm thick paraffin-embedded tissue sections. A variety of antibodies at various concentrations were selected to target nicotinic acetylcholine receptors, synaptic vesicles, and neurons. Results: Neurofilament (NEFL, Invitrogen, 1:500) and synaptic vesicle glycoprotein (SV2, DSHB, 1:230) bound and demarcated the neurons and synaptic vesicles, respectively. Following consistent visualization of nerve tissue, rabbit anti-nicotinic acetylcholine receptor alpha-1 subunit (CHRNα1, Abcam, 1:500) was used to identify the acetylcholine receptors within motor endplates. Complete NMJ visualization was then achieved with an optimized protocol using primary antibodies to the neurofilament light chain, nerve synaptic vesicle glycoprotein 2, and the alpha 1 subunit of the nicotinic acetylcholine receptor. Slide imaging was performed with the Echo Revolve Microscope (40×). Conclusions: Herein, we describe a new methodology to visualize NMJs within paraffin-embedded TA muscle sections. Our protocol avoids the known limitations associated with cryosectioned samples and introduces a new neurolaryngologic research tool that utilizes the advantageous ability of paraffin-embedded sectioning to preserve tissue morphology. In conjunction with standard cryosectioned methods, the described paraffin-embedded protocol serves to enhance histological analysis of NMJs.
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    Optimizing transport methods to preserve function of self‐innervating muscle cells for laryngeal injection
    (Wiley, 2024-12-08) Kaefer, Samuel L.; Zhang, Lujuan; Brookes, Sarah; Morrison, Rachel A.; Voytik-Harbin, Sherry; Halum, Stacey; Otolaryngology -- Head and Neck Surgery, School of Medicine
    Objectives: Recently, our laboratory has discovered a self-innervating population of muscle cells, called motor endplate-expressing cells (MEEs). The cells innately release a wide variety of neurotrophic factors into the microenvironment promoting innervation when used as an injectable treatment. Unlike other stem cells, the therapeutic potential of MEEs is dependent on the cells' ability to maintain phenotypical cell surface proteins in particular motor endplates (MEPs). The goal of this study is to identify transport conditions that preserve MEE viability and self-innervating function. Methods: Muscle progenitor cells (MPCs) of adult Yucatan pigs were cultured and induced to generate MEEs. Effects of short-term cryopreservation methods were studied on MPC and MEE stages. A minimally supplemented medium was investigated for suspension-mediated transport, and MEEs were loaded at a constant concentration (1 × 107 cells/mL) into plastic syringes. Samples were subjected to varying temperatures (4, 22, and 37°C) and durations (6, 18, 24, and 48 h), which was followed by statistical analysis of viability. Transport conditions maintaining viability acceptable for cellular therapy were examined for apoptosis rates and expression of desired myogenic, neurotrophic, neuromuscular junction, and angiogenic genes. Results: Cryopreservation proved detrimental to our cell population. However, a minimally supplemented medium, theoretically safe for injection, was identified. Transport temperature and duration impacted cell viability, with warmer temperatures leading to faster death rates prior to the end of the study. Transport conditions did not appear to affect apoptotic rate. Any expression change of desirable genes fell within the acceptable range. Conclusions: Transport state, medium, duration, and temperature must be considered during the transport of injectable muscle cells as they can affect cell viability and expression of integral genes. These described factors are integral in the planning of general cell transport and may prove equally important when the cell population utilized for laryngeal injection is derived from a patient's own initial muscle biopsy.
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    Point-of-Care Ultrasound in the Primary Care Office for Early Detection of Squamous Cell Carcinoma of the Supraglottic Larynx: A Case Report
    (Springer Nature, 2024-07-29) Wilcox, James; Kaefer, Samuel L.; Medicine, School of Medicine
    Point-of-care ultrasound (PoCUS) in the head and neck region is still in its early stages, despite decades of formal ultrasound use. A literature gap exists as recent studies primarily focus on general techniques, leaving room for exploration in ambulatory primary care, especially regarding neck masses. Our case demonstrates a 61-year-old female who presented for an annual wellness appointment, reporting a cough and alarming neck symptoms. A prompt investigation using PoCUS identified a suspicious neck mass. This mass was evaluated with bedside POCUS, and the mass was determined to be an abnormal lymph node with findings concerning malignancy. Subsequent diagnostic measures confirmed metastatic squamous cell carcinoma in the supraglottic larynx. This case underscores PoCUS's transformative potential in ambulatory primary care for neck mass evaluation, facilitating swift and thorough diagnostic processes. This successful outcome emphasizes PoCUS's promising role in routine clinical practice, urging future research for standardized evaluation protocols to enhance diagnostic efficiency.